CSIR-National Institute for Interdisciplinary Science & Technology (NIIST), Chemical Sciences & Technology Division (CSTD), Organic Chemistry Section, Thiruvananthapuram 695019, Kerala, India.
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
Anal Chem. 2021 Aug 17;93(32):11140-11150. doi: 10.1021/acs.analchem.1c01607. Epub 2021 Aug 4.
Ultrasensitive detection of cancer biomarkers via single-cell analysis through Raman imaging is an impending approach that modulates the possibility of early diagnosis. Cervical cancer is one such type that can be monitored for a sufficiently long period toward invasive cancer phenotype. Herein, we report a surface-enhanced Raman scattering (SERS) nanotag (SERS-tag) for the simultaneous detection of p16/K-i67, a dual biomarker persisting in the progression of squamous cell carcinoma of human cervix. A nanoflower-shaped SERS-tag, constituted of hybrid gold nanostar with silver tips to achieve maximum fingerprint enhancement from the incorporated reporter molecule, was further functionalized with the cocktail monoclonal antibodies against p16/K-i67. The recognition by the SERS-tag was first validated in cervical squamous cell carcinoma cell line SiHa as a foot-step study and subsequently implemented to different grades of clinically confirmed exfoliated cells including normal cell (NC), high-grade intra-epithelial lesion (HC), and squamous cell carcinoma (CC) samples of the cervix. Precise Raman mapped images were constituted based on the average intensity gradient of the signature Raman peaks arising from different grades of exfoliated cells. We observed a distinct intensity hike of around 10-fold in the single dysplastic HC and CC samples in comparison to NC specimen, which clearly justify the prevalence of p16/Ki-67. The synthesized probe is able to map the abnormal cells within 20 min with high reproducibility and stability for 1 mm × 1 mm mapping area with good contrast. Amidst the challenges in Raman image-guided modality, the technique was further complemented with the gold standard immunocytochemistry (ICC) dual staining analysis. Even though both are time-consuming techniques, tedious steps can be avoided and real-time readout can be achieved using the SERS mapping unlike immunocytochemistry technique. Therefore, the newly developed Raman image-guided SERS imaging emphasizes the approach of uplifting of SERS in practical utility with further improvement for clinical applications for cervical cancer detection in future.
通过拉曼成像对单细胞进行分析，实现对癌症生物标志物的超灵敏检测，是一种即将出现的方法，可以提高早期诊断的可能性。宫颈癌就是其中一种，可以对其进行足够长时间的监测，以观察其是否向侵袭性癌症表型发展。在此，我们报告了一种表面增强拉曼散射（SERS）纳米标签（SERS-tag），用于同时检测 p16/K-i67，这是一种持续存在于人类宫颈癌鳞状细胞癌进展过程中的双重生物标志物。纳米花状 SERS-tag 由具有银尖端的混合金纳米星组成，以从所包含的报告分子中获得最大的指纹增强，然后进一步用针对 p16/K-i67 的鸡尾酒单克隆抗体进行功能化。SERS-tag 的识别首先在宫颈癌鳞状细胞癌细胞系 SiHa 中进行了验证，作为一个前期研究，随后在不同等级的临床确认的脱落细胞中进行了实施，包括正常细胞（NC）、高级上皮内病变（HC）和宫颈癌（CC）样本。基于不同等级脱落细胞产生的特征拉曼峰的平均强度梯度，构成了精确的拉曼映射图像。我们观察到，与 NC 标本相比，单个发育不良的 HC 和 CC 标本的强度明显提高了约 10 倍，这清楚地证明了 p16/Ki-67 的普遍性。该合成探针能够在 20 分钟内对异常细胞进行成像，具有高重复性和稳定性，对于 1mm×1mm 的成像区域具有良好的对比度。在拉曼图像引导模式存在的挑战中，该技术还与金标准免疫细胞化学（ICC）双重染色分析相结合。尽管这两种技术都很耗时，但与免疫细胞化学技术不同，可以避免繁琐的步骤，并实现实时读数。因此，新开发的拉曼图像引导 SERS 成像强调了在实际应用中提升 SERS 的方法，并进一步改进了未来用于宫颈癌检测的临床应用。
